In 1993, in the United States alone, 378,000 patients were hospitalized for deep venous thrombosis (DVT) and 103,000 for pulmonary embolism (PE) (Vital and Health Statistics. Series 13: Data from National Health Survey Ditts Publication No. (PHS) 95-1783, 1993). Autopsy data suggests that the incidence of DVT and PE may be as high as 30% to 60% in the general population. For high risk patients, such as those over forty years of age or those who have undergone a major surgical procedure, the incidence of thromboembolism may be as high as 80%. (Koblik, P. D. et al., "Current status of immunoscintigraphy in the detection of thrombosis and thromboembolism", Seminars in Nucl. Med. XIX: 221-231, 1989)
In surgical patients, the initial vessel damage exposes subendothelial structures to the blood stream and the blood platelets begin to adhere to the injury site. Coagulation proteins are then activated sequentially to generate the enzyme thrombin. Thrombin cleaves plasma fibrinogen into fibrin monomers, which in turn polymerize around the clumped platelets to form a clot. Blood clots are also formed during stasis in patients confined to bed, or in those with increased intra-abdominal pressure (e.g., during pregnancy, when blood flow slows in the leg veins). Under these hemodynamic conditions, especially near venous valves or bifurcations, the coagulation factors are more likely to be activated. Many of the clots formed under these conditions are likely to be large and frequently form lethal pulmonary emboli. Arterial clots are considered to be the leading cause in the pathogenesis of myocardial ischemia or infarction and stroke.
A variety of diagnostic tests are currently available to detect DVT. These include contrast angiography (venography), radionuclide angiography, Doppler ultrasonography, thermography and impedance plethysmography. Most popular among them are the venography and ultrasonography. However, to be effective, venography and ultrasonography must be performed in the specific anatomic area of the suspected emboli. Notwithstanding this limitation, contrast venography is widely considered as the gold standard for the diagnosis of thromboembolism. Additionally, venography is an invasive technique, and the procedure itself can induce venous thrombi in patients at risk. Anticoagulant therapy to prevent formation of such venous thrombi or to lyse the existing DVT is also associated with a significant morbidity. For these reasons, a more specific and non-invasive method for imaging thrombi in the body is highly desirable. An external scintigraphy technique, aided by the use of a radiopharmaceutical, would enable a clinician to quickly scan a patient without unreasonable inconvenience or added morbidity.
In recent years, a large number of radiopharmaceuticals have been investigated as potential agents to localize DVT or PE. In that thrombi are largely composed of fibrin, platelets and other entrapped cells in the fibrin network, attention has been focused on the use of radioiodine labeled fibrinogen and In-111 labeled platelets (Thakur, M. L. et al. Thrombosis Research 1976, 9, 345-354). Antibodies specific for the fibrin binding IIb/IIa glycoprotein complex on the platelet surface have also been investigated (Thakur, M. L. Thrombotic and Hematologic Disorders 1992, 5, 29-36; Thakur, M. L. "Radiolabeled monoclonal antibodies for imaging and therapy", S. C. Srivastava (Ed.), Plenum Publishing Co., NATO ASI, series 152, 1988; Knight, L. C. Seminars in Nucl. Med. 1990, XX: 52-67. Success of these approaches has been limited due to a lack of specificity, unfavorable pharmacokinetics or cumbersome preparation of the agent.
In that platelets are a major component and a biologically active constituent of a thrombus, radiolabeling of platelets has also been considered as a diagnostic agent. However, the use of radiolabeled platelets has been less than desirable because of their long life span (roughly 8 days) that results in excessive background radioactivity well after their administration. The excessive background radioactivity causes a delay in diagnosis due to suboptimal lesion to background radioactivity ratios. Use of radiolabeled platelets is also limited by the need to prepare them in vitro by skilled personnel (Thakur, M. L., 1976, supra).
Prompted by advancements in molecular biology, radioactive agents for the non-invasive diagnosis of thromboembolism have been developed which include technetium (Tc-99m) labeled peptides specific for resting or activated platelets (Knight, L. C. et al. J. Nucl. Med. 1994, 35, 282-288; Pearson, D. A. J. Med. Chem. 1996, 39, 1372-1382; Line, B. R. et al. J. Nucl. Med. 1996, 37, 117P). Use of a peptide specifically binding to the platelet GPIIb/IIIa receptor has also been reported for imaging thrombi (Dean, R. et al., WO/94/23758).
Peptides are particularly attractive for use as radioimaging agents because they are smaller in size and easier to produce than monoclonal antibodies. Radiolabeled peptides typically clear more rapidly from circulation than radiolabeled proteins, are less likely to induce an immunological reaction, yet have equal or higher receptor specificity and binding constants than monoclonal antibodies. While a variety of radioactive nuclides have been considered for radiolabeling peptides, including Tc-99m and In-111, Tc-99m is a preferred radiolabel because of its low cost, availability, excellent imaging properties (emits gamma radiation at 140 keV), and high specific activity. Its half-life of 6 hours is long enough to perform an examination before excessive radioactive decay has occurred, yet not so long as to persist in the body long after the examination has been completed or to impart an excessive radiation dose to the patient.
Radiopharmaceuticals comprised of radiolabeled peptides and proteins can be prepared by three routes. One such route includes the use of a chelating agent such as that disclosed in U.S. Pat. No. 5,552,525. A second method utilizes a bifunctional chelating agents, such as that disclosed in U.S. Pat. No. 5,218,128. Typically, the third method, known as a direct labeling method, has been used for labeling antibodies and peptides and involves the generation of free sulfhydryl groups by the reduction of disulfide bridges to which the radionuclide, such as technetium, is chelated. See, for example, U.S. Pat. Nos. 4,917,878 and 5,011,676. The direct labeling method suffers from two prominent limitations, namely, the unstable complexes formed by technetium with the peptide or protein, and the poor control over the labeling site with the protein. It is not likely that direct labeling can be readily extended for use with small peptides because small peptides may not contain cysteine residues that are cyclized and the biological function of a small peptide may be more often altered by random addition of the radiolabel.
Bifunctional chelates have been developed which utilize high affinity chelates to bind a technetium radionuclide to specific sites on the peptide. In this latter method the chelating agent is covalently attached to the peptide prior to radiolabeling, which upon the addition of the radionuclide would result in a single radiolabeled product. This approach appears to overcome some of the limitations of the direct method of labeling in that the bioactivity and receptor-binding characteristics of the conjugate can be determined before and after labeling.
Thrombospondin (TSP), is a large (450 Kda), trimeric adhesive glycoprotein first identified as a thrombin sensitive protein more than a quarter of a century ago (Baenziger, N. L. et al. Proc. Natl. Acad. Sci. USA 1971, 68, 240-243; Ganguly, P. J. Biol. Chem. 1971, 246, 4286-4290). TSP is predominantly found in platelet a-granules and comprises nearly 3% of the total amount of platelet protein. Many different cells produce TSP, including endothelial cells, fibroblasts, macrophages and monocytes, and tumor cells. It is known that most of TSP is bound to extracellular matrix or associated strongly with basement membrane. TSP promotes cell-cell and cell-matrix interactions of normal and malignant cells, as well as platelet aggregation, and mediates angiogenesis.
TSP is stored in resting platelets in .alpha.-granules alongside other multi-functional glycoproteins such as fibronectin, fibrinogen and von Willebrand's factor. Upon activation, platelets release .alpha.-granule proteins including TSP, which then binds to the surface of activated platelets. Receptors for TSP binding on platelet surface include CD36, a 88 kd glycoprotein, GPIV and GPIIIb. One theory proposes that TSP binds to CD36 as well as to fibrinogen that is already bound to GP IIb/IIIa complex. TSP is found in fibrin meshwork of whole blood clots and excised wounds. Early wounds stain intensely for TSP whereas healed wounds hardly stain.
The structure of human TSP was determined in 1986 (Lawler et al. Blood 1986, 67, 1197-1209). It was found that the sequence Trp Ser Pro Cys Ser Val Thr Cys Gly (SEQ ID NO: 1) was present in three homologous copies in TSP, and that the sequence is a functional component in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation and tumor cell metastasis (Tuszynski, G. P. et al. J. Cell Biol. 1992, 116, 209-217).
It has been reported that human dermal microvascular endothelial cells bind to immobilized TSP via an Arg Gly Asp Cys Ser Val Thr Cys Gly sequence (SEQ ID NO: 2) (Chen et al. J. Invt. Dermatol. 1996, 106, 215-220), and that a radioiodinated (I-125) Tyr Cys Ser Val Thr Cys Gly sequence (SEQ ID NO: 3) also binds strongly to CD36 transfected Jurkat cells (Asch et al. Biochem. Biophy. Res. Comm. 1992, 182, 1208-1217). It has also been demonstrated that antibodies against TSP receptor blocked the uptake of TSP as well as that of the sequence, Cys Ser Val Thr Cys Gly (SEQ ID NO: 4) (Wang et al. Am. J. Surg. 1995, 170, 502-505).
Derivatives of the sequence Cys Ser Val Thr Cys Gly (SEQ ID NO: 4), have also been shown to have biological activity. For example, a cyclic form in which two cysteine residues are cyclized to form a five membered ring structure, and a derivative in which the two cysteine residues are blocked by acetaminomethyl (Acm), promoted binding to four tumor cell lines, approximately to the same extent as the native sequence (SEQ ID NO: 4) (Tuszynski et al. J. Cell. Biol. 1992, 116, 209-217). The native sequence (SEQ ID NO: 4), as well as the cyclic and cysteine protected forms, inhibited ADP-induced platelet aggregation, while control peptides, Ala Asn Lys His Tyr Phe (SEQ ID NO: 5) and Val Cys Thr Gly Ser Cys (SEQ ID NO: 6) displayed no biological activity.
It has now been found that a modification of the peptide sequence, Cys Ser Val Thr Cys Gly (SEQ ID NO: 4) can be used to image arterial and venous thrombi, pulmonary embolisms, and lesions of atherosclerosis. Unlike other peptides currently being investigated for use in imaging thrombi, such as, snake venom factors (Knight, L. C. et al. J. Nucl. Med. 1994, 35, 282-288), anti-platelet factor IV (Pearson, D. A. et al. J. Med. Chem. 1996, 39, 1372-1382), and the DuPont peptide DMP444 (Line, B. R. et al. J. Nucl. Med. 1996, 37, 117P), the peptide, Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 4) is an integral part of a protein that exists naturally in the molecular domain of thrombospondin, and one which appears to play a role in the formation of thrombi via activated platelets.